Michael D. Jones, Sean M. McCarthy, and Paula Hong, Waters Corp, 34 Maple Street, Milford, MA, USAJames McKearin, Prime Organics Inc., 25 Olympia Ave, Woburn, MA, USA

Figure 1. A single reaction step from the rosuvastatin synthesis reaction scheme.

GOAL

To demonstrate the benefit of different

selectivity of UPC2 compared to LC when used

for the analysis of synthetic reaction mixtures.

BAC KG ROU N D

During the development of new chemical

entities (NCE), there is a need to analyze

reaction mixtures, synthetic intermediates, and

isolated compounds. Understanding the purity

of the materials used in a reaction provides

information that is used to aid reaction rates,

inhibit catalyst quenching mechanisms, and

provide insight to scalability.

Due to the complexity of reaction mixtures,

a single chromatographic method or

technique is not sufficient for providing the

information scientists require. For example,

during the synthesis of a molecule, lack of

chromatographic selectivity can lead to

poorly separated impurities that often affect

the target molecule yield throughout the

synthetic pathway.

In terms of high-throughput screening (HTS)

using LC, screening strategies including

high /low pH have recently been implemented

to alter chromatographic selectivity. However,

this reaction monitoring process may still be

inefficient in separating structurally similar

components generated during the synthetic

process. It is critical when submitting an NCE to

a compound library that identity and purity are

assessed, thus, making orthogonal separations

strategies important.

Alternative chromatographic selectivity with UPC2

allows for robust and accurate characterization of

synthetic reaction mixtures.

Importance of Selectivity for Reaction Monitoring

SM1 SM2 Product

O

CO2CH3

OtBDMSi

Ph3P

N

N

CH3

H3CN

F

H3C

SO2CH3

O

CO2CH3

OtBDMSi

N

N

CH3

OCH

H3CN

F

H3C

SO2CH3

ACN / 14 hrsColumn

T H E SO LU T IO N

The primary aspects of this investigation evaluate Convergence

Chromatography for monitoring the total synthesis of the pharmaceutical

drug substance rosuvastatin, an HMG-CoA inhibitor. We used an ACQUITY

UPC2™ System configured with an ACQUITY UPC2 PDA for UV detection and an

ACQUITY® QDa™ Detector for mass detection. For simplicity, we compared the

ACQUITY UPC2 results from one of the synthetic stages to the results acquired

with reversed-phase UPLC® using high/low pH screening. The details of the

synthetic step are shown in Figure 1.

Figure 2. Chromatographic overlays at 270 nm of (A) ACQUITY UPC2, (B) UPLC low pH, and (C) UPLC high pH results for the initial time point (T=0 min). See figure 1 for SM1 and SM2 structures.

Figure 3. Chromatographic overlays at 298 nm of (A) ACQUITY UPC2, (B) UPLC low pH, and (C) UPLC high pH results for time point 5 (T5 = 6hrs).

AU

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-1

3.0e-1

AU

0.0

5.0e-2

1.0e-1

1.5e-1

2.0e-1

2.5e-1

-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

AU

0.0

2.0e-2

4.0e-2

6.0e-2

0.53

0.01

1.77

1.04

0.92

0.19 1.26

1.05

1.17

SM1 = m/z 352 DaSM2 = m/z 535 DaSM2

SM1

SM2

SM2

1.5 min

2.5 min

1.5 min

N

N

CH3

OCH

H3CN

F

H3C

SO2CH3

O

CO2CH3

OtBDMSi

Ph3P

UPC2

Low pH UPLC

High pH UPLC

0.0

1.0e-2

2.0e-2

3.0e-2

4.0e-2

0.0

1.0e-2

2.0e-2

3.0e-2

4.0e-2

-0.00 0.20 0.40 0.50 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

-0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00

0.0

1.0e-2

2.0e-2

3.0e-2

4.0e-2

5.0e-20.69

0.53

0.01

1.76

1.04

0.190.21

1.08

1.05

0.950.20

1.07

1.25

SM1SM2

PDT

PDT + SM1Co-Elution

SM2

SM2

AU

AU

AU

2.5 min

1.5 min

1.5 min

N

N

CH3

H3CN

F

H3C

SO2CH3

O

CO2CH3

OtBDMSi

UPC2

Low pH UPLC

High pH UPLC

Differences in chromatographic selectivity were

clearly apparent as shown in Figures 2 and 3.

At T= 0 the ACQUITY UPC2 (A), UPLC Low pH (B) and

UPLC High pH (C) separations were able to resolve

each of starting materials in the reaction mixture

(Figure 2). The reaction was monitored using both

techniques after several hours at reflux. The UPC2®

separation resolved both the starting materials

and the final product in the mixture (Figure 3). In

contrast, neither of the UPLC separations provided

enough specificity between the product (PDT) and

starting material (SM1). Additionally, the starting

material labeled “SM2” appeared quenched in both

UPLC separations, but not in the UPC2 results.

The monitoring wavelength of 270 nm was suitable

for the starting materials; however, 270 nm was not

suitable for monitoring the product material during

the UPLC analysis. The systems were configured to

acquire a 3D PDA MaxPlot data channel ranging from

210 nm to 410 nm. This capability allows for UV

spectral analysis and selected wavelength extracted

chromatograms. The PDA spectra of the product

indicated a lambda max of 295 nm. Therefore,

chromatograms were extracted from the MaxPlot

data channel to best monitor the reactions without

having to change methods prior to next experiments.

Interestingly, the SM2 material is not as easily

detected by UPLC at 295 nm. The UV absorbance

of the mobile phase interferes with the detection of

the SM2, hence appearing as a quenched reaction.

Although changing the wavelength was needed

to monitor the production of the target molecule,

the PDA 3D data collection (MaxPlot) provided the

ability to view the results at 270 nm. Deriving a

chromatogram at 270 nm combined with the MS data

enabled us to determine the presence of SM2 in the

clean-up stages of the final material.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, The Science of What’s Possible, ACQUITY, UPLC, and UPC2 are registered trademarks of Waters Corporation. ACQUITY UPC2 is a trademark of Waters Corporation. All other trademarks are the property of their respective owners.

©2014 Waters Corporation. Produced in the U.S.A. April 2014 720005020EN TC- PDF

SUMMA RY

ACQUITY UPC2 for the analysis of reaction mixtures provides added selectivity and orthogonality when

compared to reversed-phase LC high/low pH screening separations. Extracted wavelengths from the PDA

Maxplot provided the ability to monitor reactions at various wavelengths and verify UV spectral profiles

without having numerous data files to investigate. In addition, using both MS and PDA detection for either

technique provides an accurate and quantitative assessment of the reaction progress and purity.